Malaria is a disease caused by infection with parasites of the phylum Apicomplexa protozoan, namely parasites of the genus Plasmodium, globally causing more than 200 million new infections and 700 thousand deaths every year. Malaria is especially a serious problem in Africa, where one in every five (20%) childhood deaths is due to the effects of the disease. An African child has on average between 1.6 and 5.4 episodes of malaria fever each year.
Malarial diseases in humans are caused by five species of the Plasmodium parasite: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi, wherein the most prevalent being Plasmodium falciparum and Plasmodium vivax. Malaria caused by Plasmodium falciparum (also called malignantor malaria, falciparum malaria or malaria tropica) is the most dangerous form of malaria, with the highest rates of complications and mortality. Almost all malarial deaths are caused by P. falciparum. 
Briefly, the plasmodial life cycle (FIG. 1) in man starts with the inoculation of a few sporozoites through the bite of an Anopheles mosquito. Within minutes, sporozoites invade the hepatocyte and start their development, multiplying by schizogony (liver stage or pre-erythrocytic stage). After a period of 5-14 days—depending on the plasmodial species—schizonts develop into thousands of merozoites that are freed into the bloodstream and invade the red blood cells (RBCs), initiating the blood stage. In the RBC, each merozoite develops into a trophozoite that matures and divides, generating a schizont that, after fully matured, gives rise to up to 32 merozoites within 42-72 h, depending on the plasmodial species. The merozoites, released into the bloodstream, will invade other RBC, maintaining the cycle. Some merozoites, after invading a RBC, develop into sexual forms—the male or female gametocytes which also enter the bloodstream after maturation and erythrocyte rupture. If a female Anopheles mosquito takes its blood meal and ingests the gametocytes, it will become infected and initiates the sexual stage of the Plasmodium life cycle. In the mosquito gut, the male gametocyte fuses with the female gametocyte, forming the ookinete, which binds to and passes through the gut wall, remains attached to its external face and transforms into the oocyst. The oocyst will divide by sporogony, giving rise to thousands of sporozoites that are released in the body cavity of the mosquito and eventually migrate to its salivary gland, where they will maturate, becoming capable of starting a new infection in humans when the mosquito bites the host for a blood meal.
Resistance of Plasmodium falciparum to the existing anti-malarial drug chloroquine emerged in the sixties and has been spreading since then. In addition, the malaria parasite has developed resistance to most other anti-malarial drugs over the past decades. This poses a major threat to public health in tropical countries and to travellers. There is every reason to believe that the prevalence and degree of anti-malarial drug resistance will continue to increase. The growing number of insecticide resistant vectors and drug resistant parasites further increases the demand for an effective malaria vaccine. Malaria vaccines are not limited to a single mode of action and hold the potential to dramatically alleviate the burden of malaria.
Some of the difficulties to develop an efficient malaria vaccine result from the multi-stage life cycle of the parasite. Each stage of the parasite development is characterized by different sets of surface antigens, eliciting different types of immune responses. Despite the large variety of displayed surface antigens, the immune response against them is often ineffective. One of the reasons is the extensive sequence polymorphism of plasmodial antigens, which facilitates the immune evasion of the different isolates.
A pre-erythrocytic vaccine would protect against the infectious form (sporozoite) injected by a mosquito and thereby inhibit parasite development in the liver. In a previously unexposed individual, if a few parasites were to escape the immune defences induced by a pre-erythrocytic vaccine, they would eventually enter the blood-stage, multiply within the erythrocytes and establish a full-blown disease.
An erythrocytic or blood-stage vaccine would inhibit the invasion and multiplication of the parasite in the red blood cells, thus preventing (or diminishing) severe disease symptoms during the blood infection. However, it is unlikely to completely interrupt the Plasmodium life cycle and prevent transmission of the parasite by this approach.
A sexual-stage vaccine would not protect the person being vaccinated, but instead interrupt the cycle of transmission by inhibiting the development of parasites once they are ingested by the mosquito along with antibodies produced in response to the vaccine. Transmission-blocking vaccines could be involved as part of a multi-faceted strategy directed towards parasite elimination and at the same time towards prevention of parasite resistance to anti pre-erythrocytic or erythrocytic treatment.
The above-mentioned complex multistage life cycle of malaria parasites presents unique challenges for a synergistic vaccine approach. Immunity against malaria parasites is stage dependent and species dependent. Many malaria researchers and textbook descriptions believe and conclude that a single-antigen vaccine representing only one stage of the life cycle will not be sufficient and that a multiantigen, multistage vaccine that targets different, that is at least two, stages of parasite development is necessary to induce effective immunity (Mahajan, Berzofsky et al. 2010). The construction of a multiantigen vaccine (with the aim of covering different parasite stages and increasing the breadth of the vaccine-induced immune responses to try to circumvent potential Plasmodium falciparum escape mutants) can be achieved by either genetically linking (full-size) antigens together, by a mixture of recombinant proteins or by synthetic-peptide-based (15-25-mer), chemically synthesized vaccines containing several peptides derived from different parasite proteins and stages.
A single fusion protein approach being comprised of several different antigens or several different alleles of a single antigen (to induce antibodies with synergistic activities against the parasite) is hindered by antigenic diversity and the capacity of P. falciparum for immune evasion (Richards, Beeson, 2009). A large number of antigens have been evaluated as potential vaccine candidates, but most clinical trials have not shown significant impact on preventing clinical malaria although some of them have shown to reduce parasite growth. The size of the resulting fusion protein/vaccine candidate is another limiting factor allowing only the combination of a few selected antigens, not excluding that the chosen antigens are not targets of natural immunity and/or exhibit significant genetic polymorphism. Highly variable antigens with multiple alleles are obviously targets of the immune response under natural challenge, and vaccine studies of PfAMA1 and PfMSP2 suggest that allele-specific effects can be achieved (Schwartz, 2012). Currently only combination vaccines (being comprised of PfCSP and PfAMA1) are undergoing clinical trials which target the pre-erythrocytic and asexual blood stage of P. falciparum (Schwartz, 2012). A multiantigen vaccine candidate, neither a fusion, nor a combination approach, targeting all three life cycle main stages of Plasmodium (including the sexual stage in Anopheles mosquitos and thus blocking parasite transmission) has still not been tested in clinical trials.
Therefore the availability of novel and improved multicomponent, multi-stage vaccines against Plasmodium falciparum would be highly advantageous.